The human T cell leukemia-lymphoma viruses (HTLV) are a family of related retroviruses originally isolated from patients with T cell lymphoma and cutaneous manifestations. A particular subgroup of the family, type I, now known as HTLV-I, has been causatively linked to malignancies which share clinical and epidemiologic features with the disease called adult T-cell leukemia-lymphoma (ATL) which occur in certain regions of Japan (6-9), the Caribbean Basin (10,11) and the southwestern United States (12). There are no known endemic areas for HTLV-II and no known casual relationship between any specific disease with HTLV-II. The source of HTLV-II virus introduced into the intravenous drug users is not known. Widescale seroprevalence studies for HTLV-II have not been carried out.
HTLV-II is structurally very similar to HTLV-I. The two viruses share approximately 50% sequence homology (29). HTLV-II was isolated from one patient who had hairy cell leukemia but no casual relationship was found. The amino acid sequence of the env protein of HTLV-II is identical to that of HTLV-I for 69% of the residues, and an additional 14% of the amino acids represent conservative substitutions (30, 31). The X and pol genes are even more highly conserved than the env gene (32).
Because of the high degree of homology between HTLV-I and HTLV-II, standard testing assays by ELISA for HTLV-I based on whole viral lysate or recombinant proteins also cross react with HTLV-II. The peptides disclosed in U.S. No. 4,833,071 are also cross reactive with HTLV-II. No effective serological assay exists to distinguish between HTLV-I and HTLV-II env proteins although antigenic differences between the two viruses have been detected by neutralization of vesicular stomatitis virus pseudotypes (5). Two supplemental methods have been employed to confirm that antibodies to HTLV are present in samples that are shown to be reactive in an HTLV-I enzyme immunoassay. The Western Blot method for HTLV-I gives bands at p15, p19, p24, p28, p32, p36 and p55 for core proteins and at gp45 and gp61 for envelope proteins (32). The radioimmuno precipitation assay (RIPA) for HTLV-I gives bands for gp45 and gp61 for env proteins, p24 and p55 for core, and p40x for the X region (31). Neither tests, however, distinguish between the two viruses.
PCR has recently been used to distinguish between HTL-VI and HTLV-II. The PCR method provides definitive results (28). However, because of its exquisite sensitivity, it is subject to false positive results. Moreover, it is a very time consuming and expensive test.
Although the mechanism of transmission of HTLV-I is currently unknown, horizontal transmission of HTLV is clearly implicated by molecular and epidemiologic analyses (13, 14). HTLV seropositivity in regions endemic for ATL is elevated overall in the general population and further elevated among close family members of patients and in the recipients of blood transfusions (15, 16). HTLV-II seropositivity has been identified in intravenous drug users in the metropolitan areas of U.S.A. (27, 28).
This means that there is an urgent need for a safe, reliable and sensitive test to screen each blood sample before its inclusion in blood banks and to isolate blood donations derived from HTLV-I and/or HTLV-II infected individuals to avoid the inadvertent spread of the virus among patients who must receive blood transfusions, e.g. hemophiliacs and surgical patients.
There is an urgent need for a rapid and less expensive method to distinguish between infection with HTLV-I and HTLV-II. Since 1988, mandatory screening of all donors for HTLV-I has been performed and donors reactive for HTLV-I, as well as HIV must be notified of their results. The uncertainty as to which virus, HTLV-I or HTLV-II, is responsible for seropositivity, renders it very difficult to counsel the donors accurately about their risk for contracting ATL or a neurological complication of HTLV-I. A method for distinguishing HTLV-I from HTLV-II is also important for seroprevalence studies to define endemic areas for HTLV-II and pathogenicity studies for both viruses (33).
The complete nucleotide sequence of the HTLV-I virus was reported in 1983 (17). This report elucidated the structure of the HTLV-I virus at both the DNA level and the predicted protein level and permitted further serological studies of the different epitopes which may be present on the HTLV-I virus. The nucleotide sequence of the HTLV-II virus was reported in 1984, 1985 and 1986 (30, 31, 32).
Simultaneous to Seiki et al's report in 1983, Dr. Carl Saxinger at National Cancer Institute reported that the use of the isolated HTLV-I virus as a solid-phase immunoadsorbent for the development of an enzyme immunoassay for the detection of HTLV-I antibodies in the African population (18).
It was further reported by Samuel et al. (19) that a combined cloning and expression system in E. coli has been used to identify HTLV-I DNA encoded glycoproteins which reacted immunologically with antibodies in sera from ATL patients. HTL-VI DNA encoding the envelope protein was cleaved into fragments and inserted into an expression vector. The expression vectors were introduced into an E. coli host by transformation. One clone, designated as pKS400, produced an envelope protein product found to be suitable for use as an immunoadsorbent to screen a group of 28 coded sera. Antibodies that recognized the bacterially synthesized HTLV-I envelope protein sequences were found in all sera that had been shown to have antibodies to HTLV by an ELISA assay with disrupted virions as the antigen (18).
Slamon et al, Application No. PCT/US 85/01803, published on Mar. 27, 1986 under Publication No. W086/01834, described polypeptides associated with immunogenic sites of HTLV-I as expression products of the X region of HTLV-I, a highly conserved region location between env and the 3 LTR of the virus. The proteins, with a molecular weight of between 37 kd and 40 kd, were cloned and expressed as fusion proteins in E. coli. The resulting products were purified and used in liquid phase immunoprecipitation tests to screen sera. The results indicated an accuracy of from about 77% to 87% (20). All of the above failed to distinguish between infection by HTLV-I or HTLV-II because of the antigens used to detect the immunoreactivity.
Synthetic peptides have been used increasingly to map antigenic or immunogenic sites on the surface of proteins and as possible vaccines. The named inventor and a colleague previously have taken this approach to identify and characterize highly antigenic epitopes on the envelope proteins of HTLV and to develop sensitive and specific immunoassays for the detection of antibodies to HIV (previously designated HTLV-III) (21). See also U.S. Pat. No. 4,735,896, issued Apr. 5, 1988 and U.S. Pat. No. 4,879,212 issued Nov. 7, 1989, the contents of which are, hereby, fully incorporated by reference (22, 23). A similar approach is employed in this invention to select and identify highly antigenic epitopes in HTLV-I and HTLV-II. In selecting regions of the envelope protein for epitope analysis, several strategies were employed. First, regions that exhibited a relatively high conservation of amino acid sequence between HTLV-I and HTLV-II were sought. Second, multiple overlapping linear peptides covering whole regions of gp21, the transmembrane portion of the HTLV envelope protein (See FIG. 1), were synthesized and characterized. Third, multiple overlapping linear peptides covering the whole region of gp46, the external portion of the HTLV envelope protein (See FIG. 1), were synthesized and characterized. Three peptides, from the transmembrane portion, with the following sequences (See FIG. 2), and a mixture thereof, were found to be highly immunoreactive with sera from patients with ATL:
__________________________________________________________________________ GLDLLFWEQGGLCKALQEQC-NH2 (I) SEQ ID No.: 1 QNRRGLDLLFWEQGGLCKALQEQC-NH2 (II) SEQ ID No.: 2 NRRGLDLLFWEQGGLC-NH2 (III) SEQ ID No.: 3 __________________________________________________________________________
and three peptides, from the external portion, with the following sequences, and a mixture thereof, were also found to be highly immunoreactive with sera from patients with ATL (See FIG. 3):
__________________________________________________________________________ APPLLPHSNLDHILEPSIPWKSKLLTLVQLTLQS-NH.sub.2 (IV) SEQ ID No.: 4 SSTPLLYPSLALPAPHLTLPFNWTHCFDPQIQAIVSSPCH-NH.sub.2 (V) SEQ ID No.: 5 CFDPQIQAIVSSPCHNSLILPPFSLSPVPTLGSRSRRA-NH.sub.2 (VI) SEQ ID No.: 6 wherein: A = Ala = alanine, G = Gly = glycine, R = Arg = arginine, I = Ile = isoleucine, D = Asp = aspartic acid, F = Phe = phenylalnine, N = Asn = asparagine, S = Ser = serine, Q = Gln = glutamine, W = Trp = tryptophan E = Glu = glutamic acid, Y = Tyr = tyrosine, L = Leu = leucine, V = Val = valine, K = Lys = lysine, C = Cys = cysteine, H = His = histidine, P = Pro = proline T = Thr = threonine __________________________________________________________________________
An example of an analogue peptide corresponding to Peptide IV of HTLV-I and found in the same region of HTLV-II contains the following sequence:
__________________________________________________________________________ SPPLVHDSDLEHVLTPSTSWTTKILKFIQLTLQS-NH.sub.2 (X) SEQ ID No.: 10 __________________________________________________________________________
Peptides I, II and III were described in the parent application which has now issued as U.S. Pat. No. 4,833,071.
Assays for antibodies to HTLV-I and/or HTLV-II based upon chemically synthesized peptides show several advantages over assays utilizing whole disrupted virus or bacterially produced immunoadsorbents. The peptides can easily be synthesized in gram quantities by using automated solid-phase methods, thus providing a reproducible antigen of high integrity with consistent yields. Isolation of antigens from biological systems precludes such reproducibility. More importantly, non-specific reactivities seen in non-HTLV-I or non-HTLV-II infected individuals are likely due to the heterogeneity of the preparations used for assay. This is particularly true for assays using either whole virus or Escherichia coli-derived recombinant products as immunoadsorbents. In these processes, the major histocompatibility antigens or endogenous bacterial proteins of the host cells are frequently copurified with the desired antigen virus or protein. Since antibodies to these contaminating antigens are frequently found in normal individuals, false-positive results cannot be eliminated by using current antigen isolation processes.
The assay of the present invention thus clearly eliminates those false-positive reactions encountered in the other methods and, at the same time, shows a high sensitivity to truly positive sera by the substantially increased signal-to-noise ratio. This increased signal-to-noise ratio likely results from the purity of the immunoadsorbent. The assay of the present invention is also highly specific, in that peptide IV and its HTLV-II analogue (peptide X) are also found to be useful to distinguish between individual sera infected with HTLV-I or HTLV-II. That is to say, peptide IV preferentially detects antibodies to HTLV-I but not HTLV-II, and vice versa.
Furthermore, up to the present, no viable vaccine or method to provide protection against HTLV-I or HTLV-II infection has been reported. Utilization of deactivated virus provokes fears of contracting the disease, preventing its acceptability and use.
It is, therefore, an objective of the present invention to develop a detection or diagnostic procedure that does not require the use of the virus or lysates thereof as a test reagent.
A further objective is to develop a test procedure that is highly sensitive and accurate.
A further objective is to prepare a test reagent by chemical means. The synthetic reagent can than be used to detect the presence of antibodies to HTLV-I and/or HTLV-II in body fluids and diagnose ATL, thereby avoiding the danger of exposure to the virus or segments thereof and the unnecessary proliferation of the virus.
It is also an objective of the present invention to have a test reagent and procedure which can distinguish between HTLV-I and HTLV-II infection, to enable the medical profession to study the etiology of HTLV-II infection, the diseases caused by the HTLV-II virus, and its effect on the development of HIV infection in patients who are infected with both HIV and HTLV-II.
Another objective is to develop a vaccine which, when introduced into healthy mammals, including humans, will stimulate production of antibodies to HTLV-I, thereby providing protection against HTLV-I infection.
A further objective is to provide a non-viral immunogen which can be used in mammals for the development of monoclonal and polyclonal antibodies to HTLV-I.